Antimicrobial susceptibility testing

Fosfomycin susceptibility was determined by the agar dilution method for all isolates, and additionally by disk diffusion methods for E. coli only: as described by the Clinical Laboratory Standards Institute (CLSI since disk diffusion breakpoints have not yet been validated for other Enterobacteriaceae regarding fosfomycin.²⁶

Disk diffusion testing was performed using Mueller–Hinton agar (BBL Microbiology Systems, Cockeysville MD). For E. coli isolates, fosfomycin disks containing 200μg of fosfomycin and 50μg of glucose-6-phosphate (Sigma Aldrich, Dorset, UK) were used; for all other Enterobacteriaceae, disks containing amikacin (30μg), ampicillin (10μg), gentamicin (10μg) and ceftriaxone (30μg) (BBL Micro-biology Systems, Cockeysville MD). E. coli ATCC 25922, P. aeruginosa ATCC 27953 and S. aureus ATCC 25923 were utilised as control strains and compared to documented quality control reference ranges.²⁷ Following overnight incubation in aerobic conditions at 35°C, zone diameters were measured and interpreted using CLSI guidelines.²⁷

For susceptibility testing by agar dilution, Mueller–Hinton agar (BBL Microbiology Systems, Cockeysville MD) plates were prepared by serial dilution using fosfomycin disodium salt powder supplemented with 25μg ml−1 of glucose-6-phosphate (Sigma Aldrich, Dorset, UK).^14,28 For each isolate, 10μl of a 0.5ml McFarland suspension was inoculated onto the test plate using a transfer loop. Control strains, including E. coli ATCC 25922 and S. aureus ATCC 25923 were included in each set of tests. The inoculated plates were incubated in aerobic conditions at 35°C for 18–24 h. The MIC of each antimicrobial agent was defined as the lowest concentration that inhibited visible growth of the organism.